This invention generally pertains to the fields of medicine and medical diagnostics. In particular, this invention provides novel genes and polypeptides and methods for making and using them. Specifically, the compositions and methods of the invention are used to diagnose and treat Giant Cell Arteritis (GCA).
Giant cell arteritis (GCA) is a systemic vasculitis that is a serious and potentially blinding rheumatologic disease of the elderly. Current treatment of GCA requires systemic immunosuppression with profound morbidity in the affected elderly population. GCA is widely believed to be immune-mediated; however, the etiology and pathogenesis of this systemic vasculitis remains unidentified. Furthermore, diagnosis of GCA is difficult because it relies on a constellation of nonspecific signs and symptoms and a diagnostic arterial biopsy. Significantly, blindness may be the first symptom of GCA. Thus, if a way were found to better diagnose or even screen for early onset or predisposition for GCA at an earlier stage of the disease, many cases of blindness and many lives would be saved.
Currently, corticosteroids are critical in the treatment of giant cell arteritis; they reduce the incidence of blindness and rapidly relieve symptoms. However, the amounts of steroids (e.g., prednisone) needed are significant and not without side effects, particularly as they usually must be given over an extended period of time, usually about two years. Steroid treatment is not unformly effective and causes significant morbidity in up to 40% of patients because of hypertension, osteoporosis, infection, glucose dysregulation, fluid overload, and aseptic necrosis of the hip or shoulder. Alternative use of nonsteroidal anti-inflammatory drugs (NSAIDs) will lessen the painful symptoms, but they do not prevent the blindness or vascular problems. Accordingly, new methods of treating GCA are needed. The present invention addresses these and other needs.
The present invention provides novel compositions and methods in the screening for, diagnosis of and treatment of GCA.
The invention provides an isolated or recombinant nucleic acid comprising a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO:1 or a nucleic acid encoding a polypeptide, wherein the polypeptide has a sequence as set forth in SEQ ID NO:2; or a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO:3 or a nucleic acid encoding a polypeptide, wherein the polypeptide has a sequence as set forth in SEQ ID NO:4; or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:5 or a nucleic acid encoding a polypeptide, wherein the polypeptide has a sequence as set forth in SEQ ID NO:6; or a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO:7 or a nucleic acid encoding a polypeptide, wherein the polypeptide has a sequence as set forth in SEQ ID NO:8. In various embodiments, the sequence identity to SEQ ID NO:1 is at least 80%, 85%, 90%, 95%, and 98%; the sequence identity to SEQ ID NO:3 is at least 80%, 85%, 90%, 95%, and 98%; the sequence 8 identity to SEQ ID NO:5 is at least 900%, 95%, and 98%; and, the sequence identity to SEQ ID NO:7 is at least 80%, 85%, 90%, 95%, and 98%. The nucleic acid can also comprises a sequence as set forth in SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; or SEQ ID NO:7.
The invention also provides an isolated or recombinant nucleic acid comprising a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16; or has a sequence as set forth in SEQ ID NO:12. In alternative embodiments, the sequence identity to SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 is at least 80%, 85%, 90%, 95%, and 98%. The nucleic acid can also have a sequence as set forth in SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.
The invention provides an isolated or recombinant nucleic acid which specifically hybridizes to a nucleic acid comprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 under stringent conditions, wherein the stringent conditions include a wash step comprising a wash in 0.2xc3x97SSC at a temperature of about 65xc2x0 C. for about 15 minutes. The nucleic acid can be between about 15 and about 200 residues in length; between about 25 and about 100 residues in length; or between about 35 and about 75 residues in length.
The invention provides an expression vector comprising at least one nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a nucleic acid sequence of the invention. In the expression vector, the nucleic acid can be operably linked to the promoter in the sense orientation or the antisense orientation. Also provided is a transformed cell comprising the nucleic acids and/or expression vectors of the invention.
The invention provides a polymerase chain reaction (PCR) primer pair that can amplify a nucleic acid sequence of the invention, or a subsequence thereo, under in situ or in vitro conditions.
The invention provides an isolated or recombinantly expressed polypeptide, said polypeptide encoded by nucleic acid which specifically hybridizes to a nucleic acid comprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 under stringent conditions, wherein the stringent conditions include a wash step comprising a wash in 0.2xc3x97SSC at a temperature of about 65xc2x0 C. for about 15 minutes.
The invention provides a polypeptide encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 in any reading frame on either strand (e.g., coding strand or complementary strand); such exemplary polypeptide and peptide sequences of the invention are set forth herein.
The invention provides an isolated or recombinantly expressed polypeptide having 75% sequence identity to SEQ ID NO:2, 75% sequence identity to SEQ ID NO:4, having 85% sequence to SEQ ID NO:6 or 75% sequence identity to SEQ ID NO:8. In alternative embodiments, the polypeptide has 80%, 85%, 90%, 95%, and 98% sequence identity to an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:8; and, 90%, 95%, and 98% sequence identity to an amino acid sequence as set forth in SEQ ID NO:6. The isolated or recombinantly expressed polypeptide of the invention can have an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. The isolated or recombinantly expressed polypeptide can be between about 15 and about 200 residues in length; between about 25 and about 100 residues in length; or between about 35 and about 75 residues in length.
The invention further provides an immunogenic peptide comprising a subsequence of a polypeptide of the invention, exemplary sequences of which are provided herein. For example, the immunogenic peptide can have a sequence as set forth from about residue 1 to residue 92 of SEQ ID NO:2; about residue 1 to 124 of SEQ ID NO:4; about residue 1 to 48 of SEQ ID NO:6; or about residue 1 to 81 of SEQ ID NO:8. The invention also provides a fusion protein comprising a polypeptide, particularly an immunogenic peptide, and a heterologous sequence. The heterologous sequence can be any sequence not GCA-associated; for example, a sequence that aids in the expression, isolation/purification of the fusion protein.
The invention provides an isolated or recombinant antibody or binding fragment thereof which specifically binds to a polypeptide or peptide or an immunogenic fragment thereof, of the invention. The antibody can be a monoclonal antibody or a polyclonal antibody or binding fragment thereof. The invention further provides a hybridoma cell line comprising (e.g., producing) a monoclonal antibody of the invention.
The invention provides kits for detecting the presence of nucleic acid sequences associated with GCA (typically from, e.g., a serum, urine, tissue or biopsy sample) comprising a nucleic acid of the invention, or combinations thereof, where a nucleic acid in the sample detectably hybridizes to a nucleic acid of the invention under in situ or in vitro conditions. Also provided are kits for detecting the presence of nucleic acid sequences associated with GCA (also typically from, e.g., a serum, urine, tissue or biopsy sample) comprising an amplification primer pair that can amplify a sample nucleic acid having a sequence as set forth in claim 1, claim 5 or claim 9 under in situ or in vitro conditions. Further provided are kits for detecting the presence of polypeptide sequences associated with GCA comprising an antibody of the invention.
The invention also provides kits, e.g., ELISA kits, for detecting the presence of human antibodies associated with GCA in a sample comprising a polypeptide of the invention. The polypeptides or peptides in the kit can be immobilized. The kit can further comprise a non-human antibody or antisera that specifically binds to a human antibody under in situ or in vitro conditions. As described below, the non-human antibody in the kit can further comprise a detectable tag (e.g., an enzyme, a radionuclide, biotin, and the like, as discussed below), or the invention can comprise a second antibody capable of binding to the first non-human antibody.
The invention provides arrays (also called xe2x80x9cDNA chipsxe2x80x9d or xe2x80x9cmicroarraysxe2x80x9d) of oligonucleotide probes for the identification of GCA-associated nucleic acid in a sample. The nucleic acid in these arrays are typically immobilized on a solid support comprising, amongst other nucleic acids, a GCA-associated nucleic acid of the invention.
The invention provides methods for diagnosing or determining predisposition for GCA comprising the following steps: (a) providing an antibody that specifically binds to a polypeptide associated with GCA, wherein the antibody has the same specificity as an antibody of the invention (that binds to a GCA-associated peptide or polypeptide); or, a nucleic acid that can detectably hybridizes to a nucleic acid of the invention under in situ or in vitro conditions; (b) providing a tissue or fluid (e.g., whole blood, serum or urine) sample; (c) contacting the antibody or nucleic acid with the sample; and (d) detecting whether the antibody specifically binds to a polypeptide in the tissue or serum sample or the nucleic acid hybridizes to a nucleic acid in the tissue or serum sample; wherein the specific binding or hybridization is diagnostic for or determines a predisposition for GCA.
The invention provides methods for diagnosing or determining predisposition for GCA comprising the following steps: (a) providing a nucleic acid amplification primer pair of the invention that can amplify a GCA-associated nucleic acid under in situ or in vitro conditions; (b) providing a tissue or fluid (eg., whole blood, serum or urine) sample; (c) contacting the primer in pair with the tissue or fluid (e.g., whole blood, serum or urine) sample under amplification reaction conditions; and (d) detecting whether the primer pair has amplified a nucleic acid in the sample; wherein hybridization is diagnostic for or determines a predisposition for GCA.
The invention provides methods for diagnosing or determining predisposition for GCA comprising the following steps: (a) providing a polypeptide or peptide of the invention (a GCA-associated polypeptide); (b) providing a tissue or fluid (e.g., whole blood, serum or urine) sample; (c) contacting the sample with the polypeptide or peptide under physiologic conditions; and (d) detecting whether an antibody in the sample specifically binds to a polypeptide or peptide of step (a); wherein antibody binding is diagnostic for or determines a predisposition for GCA. In this method the detection in step (d) can be an ELISA assay or equivalent thereof, as discussed below.
The invention provides methods for isolating nucleic acid sequences associated with GCA comprising the following steps: (a) providing a first tissue sample from a tissue or fluid specimen not showing histologic or other signs of GCA and a second tissue sample from a tissue or fluid specimen showing histologic or other signs of GCA; (b) isolating the nucleic acid from both samples; (c) subtracting nucleic acid from the first sample from the second sample to isolate nucleic acid only present in the second sample, wherein the isolated nucleic acid from the second sample is associated with GCA-affected tissue and not normal tissue. This aspect of the invention can incorporate all variations and equivalents of subtractive hybridization techniques, as described below. In this method, the first and the second tissue sections can be taken from a xe2x80x9cskipxe2x80x9d lesion of a temporal artery of a GCA patient.
The invention provides methods for isolating lymphocytes involved in the pathogenesis of GCA comprising the following steps: (a) incubating a GCA-associated polypeptide or peptide of the invention with a plurality of adherent, irradiated antigen presenting cell cultures; (b) contacting a sample of isolated lymphocytes from a GCA patient with the polypeptide-incubated adherent antigen presenting cell cultures of step (a); (c) culturing the cells contacted in step (b) for sufficient time to allow for cytokine secretion or cell proliferation; and (d) detecting which cell culture comprises proliferating cells or cells secreting cytokines, wherein proliferation or secretion of cytokines indicates the isolated lymphocytes are involved in the pathogenesis of GCA. In this method, the lymphocytes can be B cells, stem cells or T cells. The cells can be cultured for about 2 to 5 days before cell proliferation or cytokine secretion is analyzed.
The invention provides methods for generating antibodies for the diagnosis or treatment of GCA comprising administering a GCA-associated polypeptide or peptide of the invention in amounts sufficient to generate an immune response. The immune response can be primarily humoral, cell-based, or a combination thereof. The GCA-associated polypeptides can be administered to non-human animals to generate non-human antibodies for diagnostic tests (they can also be administered to human Ab-gene-carrying mice to generate all-human antibodies, as discussed below). The GCA-associated polypeptides also can be administered to humans as vaccines for the treatment or prevention of GCA.
The invention provides methods for making the nucleic acids, polypeptides, and antibodies of the invention, as described herein. These can be isolated from nature, made in recombinant expression systems or can be entirely synthetic, as described herein. For example, nucleic acids within the scope of the invention can be identified and isolated using, e.g., nucleic acid hybridization techniques, including in situ hybridization, traditional techniques such as dot-blotting of cDNA or genomic libraries, or amplification techniques, such as PCR. Amplification primers can be designed directly from the GCA-associated nucleic acids described herein, or they can be modified, degenerate primers, as discussed below. Polypeptides can be identified and isolated using a variety of methods, including, e.g., analysis of ORFs from GCA-associated nucleic acids, binding with GCA-associated antibodies, wherein the antibodies can be, e.g., isolated from human patients or generated using the polypeptides or peptides of the invention, and the binding can be, e.g., in tissue samples or expression libraries. Antibodies can be made by inoculation of mammalian recipients using, e.g., the polypeptides or peptides of the invention, or, they can be isolated from immunized animals or expression libraries, e.g., phage (antibody binding site) expression libraries.
A further understanding of the nature and advantages of the present invention is realized by reference to the remaining portions of the specification, the figures and claims.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.